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Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells.

Chen YT, Stewart DB, Nelson WJ - J. Cell Biol. (1999)

Bottom Line: The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells.Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains.In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5435, USA.

ABSTRACT
The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

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Intracellular localization and size  exclusion column elution profile of E-cad8  are distinctively different from those of full-length E-cadherin. (A) Schematic diagram of  E-cad8 compared with full-length E-cadherin. The COOH-terminal 36 aa of E-cadherin are deleted in E-cad8. This deletion is  equivalent to the deletion in GP2CAD8 (Fig.  4). The KT3 epitope tag was fused at the  COOH terminus of the cytoplasmic domain of  E-cad8. EXT, extracellular domain; TM,  transmembrane domain; BB, β-catenin binding domain. Note the diagram is not to scale.  (B) Immunofluorescence staining of E-cad8  in MDCK cells using monoclonal antibody  KT3. Bar, 50 μm. (C) Elution profile of full-length E-cadherin, E-cad8, and β-catenin after Superose 6 size exclusion chromatography. Individual fractions were resolved by  SDS-PAGE and proteins were probed with a  monoclonal antibody against the extracellular domain of E-cadherin or a monoclonal  antibody against β-catenin. The numbers  given on top of the gels are the fraction numbers. Note that there are three polypeptide bands in the MDCK E-cad8/E-cadherin Western blot. E-cad8 has an electrophoretic mobility faster than that of full-length E-cadherin, which is consistent with the cytoplasmic domain deletion. Full-length E-cadherin is the  middle polypeptide band. A protein band with an electrophoretic mobility slower than that of the full-length E-cadherin probably  represents the precursor form of E-cad8. T, total cell lysate.
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Figure 12: Intracellular localization and size exclusion column elution profile of E-cad8 are distinctively different from those of full-length E-cadherin. (A) Schematic diagram of E-cad8 compared with full-length E-cadherin. The COOH-terminal 36 aa of E-cadherin are deleted in E-cad8. This deletion is equivalent to the deletion in GP2CAD8 (Fig. 4). The KT3 epitope tag was fused at the COOH terminus of the cytoplasmic domain of E-cad8. EXT, extracellular domain; TM, transmembrane domain; BB, β-catenin binding domain. Note the diagram is not to scale. (B) Immunofluorescence staining of E-cad8 in MDCK cells using monoclonal antibody KT3. Bar, 50 μm. (C) Elution profile of full-length E-cadherin, E-cad8, and β-catenin after Superose 6 size exclusion chromatography. Individual fractions were resolved by SDS-PAGE and proteins were probed with a monoclonal antibody against the extracellular domain of E-cadherin or a monoclonal antibody against β-catenin. The numbers given on top of the gels are the fraction numbers. Note that there are three polypeptide bands in the MDCK E-cad8/E-cadherin Western blot. E-cad8 has an electrophoretic mobility faster than that of full-length E-cadherin, which is consistent with the cytoplasmic domain deletion. Full-length E-cadherin is the middle polypeptide band. A protein band with an electrophoretic mobility slower than that of the full-length E-cadherin probably represents the precursor form of E-cad8. T, total cell lysate.

Mentions: Fluorescent antibody-stained cells, NBD-ceramide-stained fixed cells, or acridine orange stained living cells were observed using a Zeiss Axioplan fluorescence microscope with a 63× objective and the appropriate filter sets. Note that for observation of acridine orange staining, the normal filter set for fluorescein isothiocyanate fluorescence was reconfigured by replacing the barrier filter with a long pass band filter (590 nm). Fluorescent images of stained cells were recorded using Kodak Tri-X film (see Fig. 5 A) or Kodak Ektachrome Elite II (ASA 400) film (see Fig. 5, B–D). Photographic images were then digitized with a slide scanner (Nikon) and resized using Adobe Photoshop. Images in Fig. 5 and Fig. 12 were arranged and labeled using Canvas 5 and printed from the computer file using a dye sublimation printer.


Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells.

Chen YT, Stewart DB, Nelson WJ - J. Cell Biol. (1999)

Intracellular localization and size  exclusion column elution profile of E-cad8  are distinctively different from those of full-length E-cadherin. (A) Schematic diagram of  E-cad8 compared with full-length E-cadherin. The COOH-terminal 36 aa of E-cadherin are deleted in E-cad8. This deletion is  equivalent to the deletion in GP2CAD8 (Fig.  4). The KT3 epitope tag was fused at the  COOH terminus of the cytoplasmic domain of  E-cad8. EXT, extracellular domain; TM,  transmembrane domain; BB, β-catenin binding domain. Note the diagram is not to scale.  (B) Immunofluorescence staining of E-cad8  in MDCK cells using monoclonal antibody  KT3. Bar, 50 μm. (C) Elution profile of full-length E-cadherin, E-cad8, and β-catenin after Superose 6 size exclusion chromatography. Individual fractions were resolved by  SDS-PAGE and proteins were probed with a  monoclonal antibody against the extracellular domain of E-cadherin or a monoclonal  antibody against β-catenin. The numbers  given on top of the gels are the fraction numbers. Note that there are three polypeptide bands in the MDCK E-cad8/E-cadherin Western blot. E-cad8 has an electrophoretic mobility faster than that of full-length E-cadherin, which is consistent with the cytoplasmic domain deletion. Full-length E-cadherin is the  middle polypeptide band. A protein band with an electrophoretic mobility slower than that of the full-length E-cadherin probably  represents the precursor form of E-cad8. T, total cell lysate.
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Related In: Results  -  Collection

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Figure 12: Intracellular localization and size exclusion column elution profile of E-cad8 are distinctively different from those of full-length E-cadherin. (A) Schematic diagram of E-cad8 compared with full-length E-cadherin. The COOH-terminal 36 aa of E-cadherin are deleted in E-cad8. This deletion is equivalent to the deletion in GP2CAD8 (Fig. 4). The KT3 epitope tag was fused at the COOH terminus of the cytoplasmic domain of E-cad8. EXT, extracellular domain; TM, transmembrane domain; BB, β-catenin binding domain. Note the diagram is not to scale. (B) Immunofluorescence staining of E-cad8 in MDCK cells using monoclonal antibody KT3. Bar, 50 μm. (C) Elution profile of full-length E-cadherin, E-cad8, and β-catenin after Superose 6 size exclusion chromatography. Individual fractions were resolved by SDS-PAGE and proteins were probed with a monoclonal antibody against the extracellular domain of E-cadherin or a monoclonal antibody against β-catenin. The numbers given on top of the gels are the fraction numbers. Note that there are three polypeptide bands in the MDCK E-cad8/E-cadherin Western blot. E-cad8 has an electrophoretic mobility faster than that of full-length E-cadherin, which is consistent with the cytoplasmic domain deletion. Full-length E-cadherin is the middle polypeptide band. A protein band with an electrophoretic mobility slower than that of the full-length E-cadherin probably represents the precursor form of E-cad8. T, total cell lysate.
Mentions: Fluorescent antibody-stained cells, NBD-ceramide-stained fixed cells, or acridine orange stained living cells were observed using a Zeiss Axioplan fluorescence microscope with a 63× objective and the appropriate filter sets. Note that for observation of acridine orange staining, the normal filter set for fluorescein isothiocyanate fluorescence was reconfigured by replacing the barrier filter with a long pass band filter (590 nm). Fluorescent images of stained cells were recorded using Kodak Tri-X film (see Fig. 5 A) or Kodak Ektachrome Elite II (ASA 400) film (see Fig. 5, B–D). Photographic images were then digitized with a slide scanner (Nikon) and resized using Adobe Photoshop. Images in Fig. 5 and Fig. 12 were arranged and labeled using Canvas 5 and printed from the computer file using a dye sublimation printer.

Bottom Line: The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells.Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains.In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5435, USA.

ABSTRACT
The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

Show MeSH
Related in: MedlinePlus